Serum-free medium composed of a modified Ham's F-12 medium was found to support the long-term multiplication of human epithelial liver cells. These epithelial cells were positive for general cytokeratin expression as well as positive for cytokeratin 18 expression through four passages. In addition, human hepatocytes in primary culture transfected with the SV40 large T antigen gene formed foci within 6-8 weeks that were positive for both keratin and large T antigen expression. Conditioned medium from cultures of the transfected human liver cells was shown to cause a 30% increase in DNA synthesis of a malignant human liver cell line (HepG2).